基于JAK/STAT3通路探讨柚皮苷对人类风湿关节炎成纤维样滑膜细胞增殖与凋亡的影响及其作用机制  

Influences and mechanism of naringin on proliferation and apoptosis of human rheumatoid arthritis fibroblast like synoviocytes via JAK/STAT3 pathway

作　　者：田晓静[1] 马丽[1] 李春梅 黄佳珉[2] TIAN Xiao-jing;MA Li;LI Chun-mei;HUANG Jia-min(Laboratory Department,Changzhou Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Traditional Chinese Medicine,Changzhou 213000,China;Department of Rheumatology,Changzhou Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Traditional Chinese Medicine,Changzhou 213000,China)

机构地区：[1]南京中医药大学附属常州市中医医院检验科,常州213000 [2]南京中医药大学附属常州市中医医院风湿免疫科,常州213000

出　　处：《现代免疫学》2024年第5期430-436,共7页Current Immunology

摘　　要：基于JAK/STAT3通路探讨柚皮苷(naringin,NRG)对人类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis fibroblast-like synoviocytes,RA-FLS)增殖与凋亡的影响。分离RA患者的膝关节滑膜组织RA-FLS,免疫荧光法检查波形蛋白(vimentin)、CD90表达以鉴定细胞;将RA-FLS分为NC组(正常培养的RA-FLS)、NRG-L组(20μg/mL)、NRG-M组(40μg/mL)、NRG-H组(80μg/mL)、NRG-H+Coumermycin A1(JAK激活剂)组(80μg/mL NRG+10μmol/L Coumermycin A1)。CCK-8法检测RA-FLS增殖;Hoechst 33258染色检测细胞凋亡形态;流式细胞术检测RA-FLS凋亡;ELISA法检测RA-FLS培养上清液中IL-6、IL-1β、TNF-α水平;qRT-PCR检测RA-FLS中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、Caspase-3 mRNA水平;Western blotting检测RA-FLS中PCNA、Caspase-3、p-JAK2、p-STAT3蛋白表达。结果显示,细胞呈梭形生长,且vimentin、CD90表达呈强阳性,证实分离的细胞为RA-FLS;与NC组比较,NRG-L组、NRG-M组、NRG-H组RA-FLS OD_(450)值(24、48h)、IL-6、IL-1β、TNF-α水平、PCNA mRNA及蛋白、p-JAK2、p-STAT3蛋白表达降低,亮染细胞数量、细胞凋亡率、Caspase-3 mRNA及蛋白表达升高,且呈剂量依赖性(P<0.05);与NRG-H组比较,NRG-H+Coumermycin A1组RA-FLS D(450 nm)值(24、48h)、IL-6、IL-1β、TNF-α水平、PCNA mRNA及蛋白、p-JAK2、p-STAT3蛋白表达升高,亮染细胞数量、细胞凋亡率、Caspase-3 mRNA及蛋白表达降低(P<0.05)。该研究表明,NRG可能通过抑制JAK/STAT3通路抑制RA-FLS增殖,促进凋亡。To investigate the influences of naringin(NRG)on the proliferation and apoptosis of human rheumatoid arthritis fibroblast like synoviocytes(RA-FLS)mediated by the JAK/STAT3 pathway,RA-FLS were isolated from knee synovial tissue of RA patients and confirmed by the expressions of Vimentin and CD90 detected by immunofluorescence.RA-FLS were then divided into NC group(normally cultured RA-FLS),NRG-L group(20μg/mL),NRG-M group(40μg/mL),NRG-H group(80μg/mL),and NRG-H+Coumermycin A1(JAK activator)group(80μg/mL NRG+10μmol/L Coumermycin A1).The proliferation of RA-FLS was detected by the CCK-8 method and the morphology of apoptosis by Hoechst 33258 staining.The apoptotic rate of RA-FLS was detected by flow cytometry.The levels of IL-6,IL-1βand TNF-αin the culture supernatant of RA-FLS were detected by ELISA.The mRNA levels of proliferating cell nuclear antigen(PCNA)and Caspase-3 in RA-FLS were detected by qRT-PCR.And western blotting was used to detect the protein expressions of PCNA,Caspase-3,p-JAK2 and p-STAT3 in RA-FLS.The quality of RA-FLS isolation was confirmed by the spindle-shaped cells and strongly positive staining for vimentin and CD90.Compared to those of the NC group,the D(450 nm)value(24,48 h)and the expressions of IL-6,IL-1β,TNF-α,PCNA at both mRNA and protein levels,as well as p-JAK2,p-STAT3 protein expressions were decreased of RA-FLS in the NRG-L group,NRG-M group and NRG-H group.On the other hand,the number of bright-staining cells,the apoptosis rate,and expression levels of Caspase-3 mRNA and protein were increased,in a dose-dependent manner(P<0.05).Compared to those of the NRG-H group,the D(450 nm)value(24 h,48 h)and the levels of IL-6,IL-1β,TNF-α,PCNA at both mRNA and protein levels,as well as p-JAK2,p-STAT3 protein expression were increased in RA-FLS of NRG-H+Coumermycin A1 group,whereas the number of bright-staining cells,the apoptosis rate,Caspase-3 mRNA and protein expressions were decreased(P<0.05).Taken together,this study shows that NRG may inhibit the proliferation of RA-FLS and pr

关 键 词：柚皮苷 类风湿关节炎 滑膜成纤维细胞 Janus蛋白酪氨酸激酶/信号转导子与转录激活子3通路 增殖 凋亡 

分 类 号：R735.3[医药卫生—肿瘤]